Etrian Odyssey II requires players to slowly make their way up the trunk of a massive magical tree in order to reach a floating castle at the top while completing smaller quests along the way. The tree is represented by a series of vertically-stacked levels which are explored one at a time with frequent return trips to town in order to rest and resupply.

In classic fashion, players navigate through the forest locales a single "step" at a time, moving and turning in fixed increments. Time passes only when an action is taken, causing movement, encounters, and combat to all be entirely turn-based. While randomly generated enemies cannot be spotted in advance, certain more powerful "FOEs" usually can be seen on the map and avoided if necessary. The game uses a first-person view to present the labyrinth using a combination of relatively simple 3D computer graphics for environments and single-frame 2D sprites for enemies.

Similar to early computer role-playing games that did not support automatic mapping, players of Etrian Odyssey II must maintain their own maps. This is done by using the stylus to manually fashion maps on the DS touchscreen, with whatever level of detail is desired. Periodic warp points allow players to escape the labyrinth in order to save the game, so being able to successfully return to these (with the assistance of an accurate map) can be quite important. When compared to the mapping system in the original Etrian Odyssey, there are now additional symbols which can be used and a setting which toggles whether the map automatically follows the player or not.

Featuring no pre-made characters, the title instead requires players to create their own from twelve different character types, each with at least one special skill or aptitude. While only five characters can be in the party at once, up to thirty can be created and kept in waiting back at the "guild hall". Characters can be switched in and out when in town, so if a given specialty is needed for a specific obstacle, the party can be tailored appropriately. The player further customizes characters by allocating skill points to specific skills during level advancement.